In Vitro Cytochrome P450 Induction and Inhibition

928 Current Drug Metabolism, 2008, 9, 928-939

1389-2002/08 $55.00+.00 2008 Bentham Science Publishers Ltd.

In Vitro Cytochrome P450 Inhibition and Induction

Robert L. Walsky* and Sherri E. Boldt

Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, CT, 06340, USA

Abstract: The assessment of in vitro inhibition and induction of the cytochrome P450 enzymes of the liver is a critical part of the drug

discovery and development process in order to ensure that two or more drugs can be safely coadministered without alterations in expo-sure. Early assessment of potential candidates using high throughput approaches provides key direction in choosing the most promising

chemical series to pursue. In later stage development, the use of in vitro data to assess the potential for clinical interactions is now a prac-tice readily accepted by regulatory authorities. Inhibition of drug metabolizing enzymes can occur via two principal mechanisms, reversi-

ble inhibition and time dependent inhibition (mechanism-based inactivation). Clinically, either of these mechanisms can lead to reduced clearance of a coadministered drug and potentially toxic levels may be reached. Inducers of a drug metabolizing enzyme can increase the

clearance of other drugs, or itself, resulting in a decreased therapeutic effect; they can also increase the bioactivation of drugs that can produce reactive intermediates, leading to hepatotoxicity. A number of in vitro models composed of human-derived microsomes, recom-

binantly expressed human drug metabolizing enzymes, human-derived cell lines, as well as fresh and cryopreserved human hepatocytes, are increasingly in use to evaluate inhibition and induction. In this review, the authors understanding of currently utilized enzyme inhibi-

tion and induction methodologies are presented and the authors provide recommendations regarding which assay types offer the greatest advantage during the drug development process.

Keywords: Time dependent inhibition, mechanism-based inactivation, inhibition, induction, cytochrome P450, mass spectrometry, in vitro, hepatocyte

INTRODUCTION

The majority of drug candidates are substrates for cytochrome P450-mediated metabolism and therefore have the potential for being the object (victim) of a drug-drug interaction (DDI). Conse-quently, drugs which can inhibit or induce cytochrome P450 me-tabolism are of great concern to scientists involved in drug research, regulatory authorities, physicians, and their patients. As polyphar-macy, or the practice of prescribing multiple drugs simultaneously for a single or multiple indications has become a more common practice, drug interactions have been cited as one of the major rea-sons for hospitalization and even death [1]. Thorough characteriza-tion using in vitro systems can guide clinical DDI studies and con-tribute to appropriate product labeling. Moreover, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions. Inhibition-mediated drug-drug interactions cause a reduction in clearance and a resulting increase in AUC of a coad-ministered drug thereby increasing the possibility of that drug reaching toxic levels. An example is the coadministration of ter-fenadine, an antihistamine, with ketoconazole leading to fatal ar-rhythmias in several patients [2]. Terfenadine is metabolized pri-marily by CYP3A, and ketoconazole is a very potent CYP3A in-hibitor (IC50 ~20 nM). Terfenadine is normally a rapidly cleared drug being almost undetectable in plasma due to high first pass metabolism and pharmacologic effect resides in its metabolite, fexofenadine. However, at higher concentrations, it can also block the delayed rectifier potassium current that controls the duration of the QT heart beat interval. The effect of terfenadine metabolism being severely inhibited during coadministration with ketoconazole led to episodes of torsade de pointes, fatal arrhythmias, and ulti-mately the withdrawal of the drug from clinical use. Enzyme inhibi-tion can occur via two main binding mechanisms, reversible or irreversible. Irreversible, or mechanism-based inactivation can re-sult when a compound is metabolized by a CYP to a reactive inter-mediate that binds to the enzyme and renders it permanently inac-tive. The enzyme activity is permanently lost and clearance is re-duced for that enzyme type. Normal clearance can only be reestab-

*Address correspondence to this author at the Pfizer Global Research and Development, Eastern Point Road, Groton, CT, 06340, Tel: (860) 715-3048:

Fax: (860) 715-7866: E-mail: [email protected]

lished by the de novo synthesis of enzyme. Mechanism based inac-tivation accounts for some of the most potent clinically observed DDIs. As an example, mibefradil, a mechanism based inactivator of CYP3A, increases the AUC of the CYP3A substrate triazolam by 9-fold [3,4]. Time dependent inhibition (TDI) is the in vitro property that is assessed to differentiate between potential mechanism-based inactivators and reversible inhibitors. As a result, the assessment of test compounds for time dependent inhibition has become much more common in the drug discovery process and is an area of active research [4-6].

Induction-mediated drug-drug interactions lead to increased clearance of a coadministered drug leading to reduced efficacy. The smooth endoplasmic reticulum (ER), where the cytochrome P450 (CYP) enzymes are located, is unusually abundant in the hepato-cyte. When large concentrations of an inducing drug enter the circu-lation, these CYP liver enzymes are synthesized in unusually large amounts in an attempt to clear the drug, and the smooth ER can double in surface area, leading to a significant cell enlargement resulting in hepatocellular hypertrophy and increased liver weight. This is primarily an adaptive response as the size of the liver in-creases in proportion to the increased functional load. This is a temporary effect; once the drug is removed, the surplus ER is re-moved from the hepatocyte by the lysosomes of the cell in an auto-phagocytic process [7]. Compared to CYP inhibition, this is a slow response; inhibition is a fraction of a second process, while induc-tion can take hourstypically, in vitro cells need to be exposed to compound for 48-72 hours in order to generate a robust induction response. An additional induction concern is the increased risk of reactive metabolite toxicity due to an induction-mediated imbalance of detoxification and activation [8]. An example of the latter would be the metabolism of acetaminophen by CYP2E1; the resulting product is a highly reactive intermediate that is detoxified by con-jugation with glutathione. Since CYP2E1 is inducible by ethanol, individuals with alcohol dependence face increased risk of aceta-minophen hepatotoxicity due to increased formation of the reactive intermediate and resulting decreased glutathione concentrations [9].

Ethanol is both a CYP2E1 inhibitor at high doses and a CYP2E1 inducer at low doses; many examples exist in the literature of the same drug exhibiting characteristics of both an inhibitor and an inducer in vitro, depending on the dose and the duration of treatment. Fahmi et al. examined 32 drugs in three in vitro assays

In Vitro Cytochrome P450 Inhibition and Induction Current Drug Metabolism, 2008, Vol. 9, No. 9 929

reversible inhibition, time-dependent inactivation, and induction [10]. Of these, 12 were positives in all three in vitro assays. The authors additionally developed a mathematical model that could incorporate all three mechanisms resulting in reasonable predictive accuracy for drugs with a net inhibition and/or a net induction in the clinic.

Drug-drug interactions caused through induction are generally less significant than those caused by enzyme inhibition or inactiva-tion due to the increased metabolism of the object (victim) drug resulting in lower exposure and generally reduced toxicity [11,12]. However, the impact of an affected drugs concentration being driven below its level of efficacy cannot be underestimated, particu-larly for indications that are life-threatening. A classic example of an induction-mediated DDI is the coadministration of cyclosporine, an immunosuppresive agent, with rifampin, an antibiotic used for treating tuberculosis. Rifampin is a potent inducer of CYP3A activ-ity and when coadministered with cyclosporine, a CYP3A sub-strate, plasma levels of cyclosporine can drop below the level of efficacy leading to an increased incidence of organ rejection [13].

While many induction-based DDI are attributed to increased CYP3A activity, exposure to an inducer of other cytochrome P450 isoforms in concurrence with drug administration can lead to a significant reduction in therapeutic efficacy. For example, induction of CYP1A2 resulting from exposure to cruciferous vegetables in the diet can result in decreased efficacy of warfarin, theophylline and clozapine [14]. Polycyclic aromatic hydrocarbons, a product of tobacco combustion pertaining to cigarette smoking, are primarily responsible for inducing CYP1A1 and CYP1A2 enzymes, which in turn convert the hydrocarbons into carcinogens [15]. CYP1A1 is primarily involved in the activation of procarcinogens [16]; this isoform is mostly extrahepatic in humans and is present in the lung and placenta [17]. CYP1A1 also activates benzo[a]pyrene, a major carcinogen found in cigarette smoke [18]. CYP1A2 is primarily a hepatic enzyme and is responsible for the metabolism of caffeine, theophylline, acetaminophen, and tacrine [19], as well as for N-oxidation of some procarcinogenic arylamines [20].

Approximately 70% of human liver CYP content relates to CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A enzymes. CYP3A is by far the most abundant CYP isoform, constituting 30% of the CYP liver enzyme total in humans; it is also substantially expressed in the intestine, and plays a dominant role in drug clearance this iso-form is responsible for approximately 60% of CYP-mediated me-tabolism of all marketed drugs [21]. The FDA draft guidance on drug interaction studies identifies all the above isoforms as requir-ing consideration during the regulatory submission process [22]. Two of these enzymes, CYP2A6 and CYP2E1, are considered mi-nor enzymes and are typically evaluated only when needed. Sub-strates of CYP3A4 comprise a diverse set of varying structures; closer study of the reactions catalyzed by this enzyme has revealed that each substrate oxidation involves both regio- and stereoselec-tivity. Two major forms of CYP3A are expressed in adult human tissues: CYP3A4 and CYP3A5. While the former is found in the liver and small intestine of most individuals, CYP3A5 is polymor-phically expressed in the liver, small intestine, kidney, and other organs of those individuals who carry the CYP3A5 allele [23]. Ap-proximately 85-95% of Caucasians, 65% of Asians, and 55% of African-Americans have no functionally active CYP3A5 [24]. Such polymorphisms can contribute to interpatient variation and may impact the clinical efficacy and safe dosing of a given drug. For example, recent studies of heart, lung, and kidney transplantation patients revealed a significant association between the CYP3A5 polymorphism and tacrolimus (which is used to prevent organ re-jection in transplant recipients) dose-adjusted blood levels [25-28].

Assessing a test compounds ability to inhibit and induce cyto-chrome P450 drug metabolizing enzymes is a critical component during both the drug discovery and development processes, how-

ever, each phase utilizes differing methodologies due to the desired end use of the data. During drug discovery many thousands of compounds may be screened and rank ordered based on their in-hibitory potential to quickly determine which candidate(s) may warrant further progression. Compounds lacking the requisite char-acteristics are routinely discarded in favor of those with the best overall properties. Since drug development considers many vari-ables, a candidate selected for development may not be completely devoid of inhibition or induction activity. Developing a screening strategy that provides fit for purpose with consideration of cycle time and cost are equally important. An overview of current inhibi-tion and induction methods follows with the advantages and disad-vantages associated with each highlighted. Details around data analysis have been included for assays which the authors believe represent the current standards for in vitro inhibition and induction practices.

GENERAL INHIBITION ASSESSMENT STRATEGY

Screening assays are typically performed at a single test com-pound concentration and the resulting activity compared with the control activity and represented as percent inhibition. Using a single test article concentration allows for simplified interpretation of results and readily lends itself to automated analysis. These assays usually contain a positive control inhibitor to assess the proper as-say function but do not include the standards, QCs, or extensive documentation that later-stage development assays require. The major enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A are generally screened at a concentration of approximately 3 M and the resulting inhibition data evaluated to determine if a compound series may possess a DDI liability that should be avoided [29]. Results are often not reported as discrete quantities but rather as being of low, moderate, or high inhibitory potential in a process frequently referred to as binning. During early discov-ery, the desire to limit the potential for drug interactions must al-ways be balanced against the potential benefit of any drug candi-date.

As compounds progress through development the need for more accurate and precise kinetic constants increases dramatically. At this point, a small number of compounds are carefully examined to provide guidance for the clinical development plan and data needs to be of very high quality. The obtained constants are frequently used in conjunction with other parameters, each with its own level of uncertainty, in modeling predictive clinical outcomes across diverse population sets [30,31]. These experimental results will define if and when a clinical drug-drug interaction study may be required during clinical development and in the case of enzymes for which these in vitro inhibition data show no relevant inhibition, a patient safety endpoint is established and no further clinical assess-ment is conducted. Since these data are also reported in regulatory submissions (IB, IND, NDA etc.) as well as appearing in the final product labeling, regulatory guidelines exist and can be found in the 2006 FDA draft guidance document Drug Interaction Studies Study Design, Data Analysis, and Implications for Dosing and La-beling.[22]. Generating these data requires a very thorough as-sessment of the underlying kinetic parameters for each substrate and each source of enzyme used (e.g., pooled human liver micro-somes, recombinant enzymes) [32,33]. Incubation times need to be demonstrated to be within the linear phase of metabolite formation during a time course study to ensure reaction velocities are accu-rate, substrate depletion must be determined to be no more than 10 20% to guard against excessive test compound depletion, accurate KM determinations need to be made to ensure appropriate substrate concentrations are being used for each experiment type (IC50, Ki, TDI, KI/kinact), and the lowest protein concentration that will yield sufficient metabolite formation should be chosen to reduce the po-tential for microsomal protein binding [34]. The analytical method-ology must be equally robust, analytical standards and QCs are employed with internal standards to minimize the effects of injector

930 Current Drug Metabolism, 2008, Vol. 9, No. 9 Walsky and Boldt

variability. An interference QC should be included for each test compound to assess its effect on the analytical system. This is pre-pared by adding the test compound to a low QC sample containing the substrate, buffer, and enzyme. Results of this QC validate the test compound does not interfere with the analytical method by enhancing or suppressing the low QC response. An additional QC is the substrate blank which identifies the lack of metabolite being present in the substrate being used. If small quantities of metabolite are found to be present in the substrate, this QC allows the back-ground subtraction of endogenous material.

General Inhibition Assay Conditions

Many incubation parameters are common to most assay types including temperature (37 C), pH ~7.4, potassium phosphate buffer (25-100 mM), and optionally, MgCl2 (~3mM) [33,35]. Incu-bations are usually initiated by the addition of NADPH (~1 mM) to the incubation and terminated by adding acid to change the pH by ~2 pH units, or by adding cold organic solvent to inactivate the enzyme(s) being examined. NADPH can be either added as a neat aqueous solution of reduced NADPH or as part of a isocitrate or glucose-6-phosphate regenerating system which generates NADPH in situ from NADP

+ using an enzymatic reaction [36,37]. Both of

these methods provide similar results. Samples are either centri-fuged or filtered using 96-well filter plates prior to analysis. Test compounds are added to incubations as concentrated stocks pre-pared in solvent. Due to the diversity of chemotypes examined dur-ing early screening, DMSO is the solvent of choice. When the as-sessment strategy permits optimization, an acetonitrile/water com-bination is preferred due to its reduced impact on substrate turn-over. [38-40]. Regardless of the dissolution vehicle used, organic solvent concentrations should be maintained below 1% v/v. Micro-somal or recombinant enzyme concentrations are usually chosen based on the detection limits of the assay method being used with lower protein concentrations being desirable to reduce the effects of microsomal protein binding which can lead to reduced test com-pound being available at the enzyme active site and a resulting de-crease in observed inhibitory potency. A general comparison of the following assay types can be found in Table 1.

Fluorescence Assays

Fluorescence or fluorogenic based assays have been the most commonly used system for assessing inhibition in a high throughput discovery setting [41,42]. These assays rely on the use of recombi-nant human P450 enzymes heterologously expressed in insect cell lines because fluorescent probes lack sufficient isoform selectivity to work well with liver microsome preparations. A nonfluorescent or low fluorescence probe substrate is metabolized by the enzyme to a more highly fluorescent metabolite and its formation in the presence and absence of test compound is monitored by a fluores-cence reader [42,43]. Incubations are normally conducted in 96- or 384-well plates with assay volumes typically below 100 L and the entire experiment carried out in situ. Since these assays do not re-

quire metabolite separation and can be read using high capacity fluorescent plate readers, data analysis is simplified and extremely high throughputs are possible. However, if test compounds are ei-ther fluorescent or fluorescence quenching at the wavelengths being monitored, the assay may be compromised. Of greater concern is the generally weak correlation of fluorogenic results with those obtained using conventional (drug) probes in human liver micro-somes. Cohen et al. showed that correlations were generally weak with a significant number of false negatives and the poorest correla-tions were found for CYP3A, the most prevalent drug metabolizing enzyme [37].

Bioluminescence Assays

Bioluminescence or luminogenic based assays have only re-cently become available [44]. They utilize derivatives of D-luciferin as substrates that release luciferin as the metabolite when incubated with specific CYP enzymes. The addition of a detection reagent containing luciferase, ATP, and a detergent stops the CYP activity and initiates a luciferase reaction which generates an amount of light proportional to the amount of luciferin metabolite generated by the CYP enzyme. Major enzymes for which there are currently substrates available include CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The format of the assay is similar to the fluorescence assay in that it typically requires the use of recombinant enzymes, does not require metabolites to be re-solved and can be read using high capacity plate readers. Biolumi-nescence assays can be used to assess fluorescent or fluorescence quenching compounds that can be problematic in the previously discussed fluorescence assays. Compounds which may inhibit lu-ciferase activity could be a source of assay error. Good correlation with single probe microsomal literature inhibition data across sev-eral major enzymes has been reported by one investigator [44].

Cocktail Microsome Assays

Cocktail assays represent a single incubation containing multi-ple CYP substrate probes (Table 2) and are becoming more preva-lent in the current literature [29,35,45]. After incubation the me-tabolites formed are resolved chromatographically and quantified by MS/MS detection. Advances in mass spectrometer detection now allow the simultaneous evaluation of many metabolites with-out a significant loss in sensitivity. While analytical methodologies are becoming widely published, there is a paucity of data concern-ing observed enzyme kinetics within such systems. Potential liabili-ties of the cocktail approach include susceptibility to ion suppres-sion and probe-probe interactions [46]. The kinetics of each enzyme evaluated are often assumed to be similar to those observed using a single probe substrate, however particular care must be taken to ensure that all the substrates used and metabolites formed do not impact the observed enzyme kinetics [47-49].

Radiometric Assays

Radiometric assays rely on 3H- or

14C radiolabeled probe sub-

strates similar to those used for single probe microsomal

Table 1. Comparison of In Vitro Assay Types

Assay Type Fluorescence Bioluminescence Single Probe

LC/MS/MS

Radiometric Cocktail LC/MS/MS

Enzyme Source Recombinant Recombinant Human derived/

Recombinant

Human derived/

Recombinant

Human derived/

Recombinant

Drug Substrate No Drug-like Yes Yes Yes

Detection Method Fluorescence Luminescence LC/MS/MS Scintillation LC/MS/MS

Potential Interferences Low Low Very Low Very Low Low-Med

Throughput High High Low Low Med

Relative Cost/Sample Low Low High Med-High Med

Regulatory Compliant No No Yes Yes No


LC/MS/MS assays [50,51]. The labeled metabolite formed is typi-cally resolved chromatographically from the labeled substrate and then quantified by radiometric detector. Other methods have util-ized the liberation of

14C formaldehyde which can be extracted and

analyzed by radioluminescence detection [52]. Because these as-says use

3H- or

14C-labeled drug substrates, they have known solu-

bility and established enzyme kinetics. However, due to the re-quirement for a separation step, and the additional training, disposal costs, and regulatory oversight the use of radiolabeled compounds require, they are not widely used.

Single Probe Microsomal LC/MS/MS Assays

Single probe microsomal LC/MS/MS assays represent the Gold Standard in assessing in vitro P450-based inhibition. When evaluating other technologies, correlation with results obtained using single probe LC/MS/MS assays are routinely performed. Typical substrates used and their isoform activities monitored are shown in Table 2. Probe substrates with well demonstrated specific-ity for a single P450 enzyme are used in an incubation containing a pool of human liver microsomes, hepatocytes, or recombinant en-zyme and the CYP-specific metabolite formed monitored using chromatographic separation of metabolite(s) from substrate fol-lowed by MS/MS detection [32,33]. This is a highly sensitive de-tection strategy which offers great specificity and minimal opportu-nity for the test article to interfere with the assay. High sensitivity has the added benefit of allowing the use of low microsomal protein concentrations during incubation which greatly reduces the effects of protein binding when assessing lipophilic compounds. The choice of LC/MS/MS detection over more conventional HPLC-UV or HPLC-fluorescence detection is due to increased selectivity, and in most instances, sensitivity of MS/MS detection. It must be rec-ognized that many of the currently utilized probe substrates were initially validated using higher microsomal protein concentrations with HPLC-UV and/or HPLC-fluorescence detection and high qual-ity data is still generated using these older technologies.

Data Analysis

IC50 determinations are typically performed using 7-10 test compound concentrations including 0 with the goal being to equally span the IC50 as shown in Fig. (1A). In practice, having at least one concentration higher, or lower, than the IC50 will usually provide

very similar results Fig. (1B) and should be considered a require-ment for regulatory submission of the result (the IC50 must lie within the concentration range tested). The assay is typically con-ducted in duplicate or triplicate with the substrate concentration at or below KM so that competitive inhibition can be readily observed. After incubation, test compound activities are compared with those containing vehicle (solvent) and a percent control activity for each test concentration is obtained. The test compound concentrations with their corresponding percent control activities are fit using a graphing program to an IC50 equation such as:

+

=50IC[I]

[I]BA100Activity Control %

in which [I] is the inhibitor concentration, the IC50 represents the inflection point, and the value of 1-(A-B) is the maximum inhibi-tion observed at an infinite inhibitor concentration. If a selective substrate is utilized, the span between A and B should approach unity. Replicates can be averaged or, depending on the graphing program used, simultaneously fit with the corresponding deviation from mean plotted. IC50 values are usually reported with a standard error and an r

2 value assigned by the fitting program. Low standard

errors (0.95) are indicative of

very good fitting characteristics.

Ki determinations are performed to identify the mechanism of inhibition of a test compound and are usually performed for regula-tory submission. A determination of Ki requires the use of multiple substrate and test compound concentrations with the resulting data fit to a non-linear plot (e.g., Eadie-Hofstee) and the mechanism, competitive, non-competitive, or uncompetitive chosen using best fit criteria (e.g., Akaike Information Criteria). A typical approach is to use four substrate concentrations spanning 3 to 5-fold above and below the substrate KM and six test compound concentrations in-cluding zero spanning 4 to 6-fold above and below the anticipated Ki. The assay is typically run with duplicate samples and, after in-cubation, the metabolite formed quantified. A rate of metabolite formation is calculated as picomoles or nanomoles of metabolite per mg of microsomal protein per minute of incubation time (nmol/mg/min) and combined with their corresponding test com-pound and substrate concentrations, entered into an enzyme kinetics curve-fitting program (e.g., SigmaPlot) and the best fit of the data

Table 2. FDA Recommended Substrate Drugs for In Vitro DDI Experiments

Enzyme Substrate Activity Metabolite Ref

CYP1A2 Phenacetin

Tacrine

Phenacetin O-deethylase

Tacrine 1-hydroxylase

Acetamidophenol

1-Hydroxytacrine

[33]

[79]

CYP2A6 Coumarin

Nicotine

Coumarin 7-hydroxylase

Nicotine C-oxidation

7-Hydroxycoumarin

Cotinine

[33]

[80]

CYP2B6 Bupropion

S-Mephenytoin

Bupropion hydroxylase

S-Mephenytoin N-demethylase

Hydroxybupropion

Nirvanol

[33]

[35]

CYP2C8 Amodiaquine

Taxol

Amodiaquine N-deethylase

Taxol 6-hydroxylase Desethylamodiaquine

6-Hydroxytaxol [33]

[81]

CYP2C9 Diclofenac

Tolbutamide

S-Warfarin

Diclofenac 4-hydroxylase

Tolbutamide hydroxylase

S-Warfarin 7-hydroxylase

4-Hydroxydiclofenac

Hydroxytolbutamide

7-Hydroxywarfarin

[33]

[33]

[82]

CYP2C19 S-Mephenytoin

Omeprazole

S-Mephenytoin 4-hydroxylase

Omeprazole 5-hydroxylase

4-Hydroxymephenytoin

5-Hydroxyomeprazole

[33]

[82]

CYP2D6 Dextromethorphan

Bufuralol

Dextromethorphan O-demethylase

Bufuralol hydroxylase

Dextrorphan

Hydroxybufuralol

[33]

[82]

CYP2E1 Chlorzoxazone Chlorzoxazone 6-hydroxylase 6-Hydroxychlorzoxazone [33]

CYP3A Felodipine

Midazolam

Nifedipine

Testosterone

Felodipine oxidase

Midazolam 1-hydroxylase

Nifedipine oxidase

Testosterone 6-hydroxylase

Dehydrofelodipine

1-Hydroxymidazolam

Dehydronifedipine

6-Hydroxytestosterone

[33]

[33]

[83]

[33]


Fig. (1). CYP2B6 Bupropion hydroxylase thioTEPA IC50 with concentra-

tions fully spanning the IC50 in panel A () and partially spanning the IC50 in panel B ().

determined by evaluating each mechanism equation (competitive, non-competitive, or uncompetitive):

Competitive Inhibition: [ ]

[ ]( ) [ ]SKIK

SVv

iM ++

=

/1

max

Noncompetitive Inhibition: [ ]

[ ]( ) [ ]( ) [ ]SKIKIK

SVv

iiM +++

=

/1/1

max

Uncompetitive Inhibition: [ ]

[ ]( ) [ ]SKIK

SVv

iM ++

=

/1

max

The best fit is determined by using the mechanism showing an optimal Akaike Information Criteria (AIC) and associated r

2 fit.

Determining the optimal concentration of test compound to perform a Ki experiment is often difficult and it is common to repeat the experiment until the Ki is adequately bracketed by the concentra-tions used. Graphically presenting the data allows for visual deter-mination of fit, however the r

2 and AIC values are needed to effec-

tively establish optimal fit of a model. Fig. (2) shows examples of Michaelis-Menten, Lineweaver-Burke, and Eadie-Hofstee plots of ketoconazole competitive inhibition.

Fig. (2). CYP3A midazolam 1-hydroxylase inhibition by ketoconazole,

Mechanism of inhibition (Ki) plots of the same dataset. Michaelis-Menten

plot (panel A), Eadie-Hoffstee plot (panel B), and Lineweaver-Burk plot

(panel C).


Time Dependent Inhibition Assays

Time Dependant Inhibition (TDI) assays are performed to de-termine: 1) If a compound may be a Mechanism Based Inactivator (MBI), and 2) if needed, a determination of the inactivation con-stants, kinact, the limit maximum inactivation rate, and KI, the con-centration at kinact. These constants can be used with other ADME parameters to attempt prediction of clinically relevant DDIs [4-6]. Recent work by Polasek and Minors have shown that the results obtained using recombinantly prepared enzyme systems sometimes show distinctly different results from those obtained using human liver microsomes [53]. One proposed theory is that differing ratios of reductase to CYP enzyme found in recombinant systems may lead to increased catalytic turnover and subsequent enzyme inactivation. In general, TDI assays utilize a preincubation step in which the test compound, NADPH and microsomes are incubated at a high concentration and after the preincubation time has been met, an aliquot is diluted into substrate and an activity assessment is made. Diluting the inhibitor and enzyme lowers the impact of reversible inhibition on the assay result. It is important that appropriate controls are included in the experiments to account for the natural loss in microsomal activity over the preincubation period. If preincubated test compounds show reduced activity over controls, this is not proof of relevant TDI. Instead it is an indication that additional, much more time consuming, KI/kinact assessments may be needed. A novel approach has been demonstrated using CYP1A2 inactivation which allows the simultaneously assessment of Ki, KI, and kinact during a single experiment [54]. This approach requires further evaluation across a broader set of enzymes and inactivators but appears promising.

Single Point and IC50-Shift TDI Assay

A relatively simple (in execution) TDI experiment is the single concentration TDI assay [4,6]. In this assay a single test compound concentration (IC25) is used to maximize to possibility of observing a significant shift in activity over controls (Fig. (3) vertical arrow). The IC25 concentration is calculated from data obtained when de-termining a reversible IC50. (if a compound does not inhibit a CYP, it is run at the highest conc. previously evaluated. Therefore this is an iterative approach and not well suited for higher throughput use. The test compound is run at 10X its IC25 conc. in microsomes at 10X their normal incubation concentration in the presence and ab-sence of NADPH. After a 30 minute preincubation, aliquots are diluted 10X into substrate at KM containing NADPH and a normal incubation conducted. At this point the test compound is at its theo-retical IC25 conc., substrate at KM, and microsomes at their normal conc. When results are compared to vehicle controls and percent control activities calculated, a direct comparison of percent change between them is made to determine if TDI is of concern or not. Typically a loss of more than 15-25% activity is considered rele-vant and further investigation is warranted. This type of experiment does not prove that the tested compound is a mechanism-based inactivator, instead it shows that time-dependent inhibition is pre-sent and that further evaluation is warranted in the form of a KI/kinact assessment. When assessing time dependent inhibition, greatest effort is usually given towards CYP3A due to the very large number of drugs it metabolizes. However, it is important to recognize that mechanism-based inactivators of CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, and CYP2E1 have been identified and that a thorough TDI assessment of all relevant en-zymes is critical during the drug development process [4].

KI/kinact Assay

Inactivation kinetics experiments are conducted similarly to the TDI experiments described above [6]. A preincubation of test com-pound at several concentrations including zero with NADPH is performed at several preincubation time points, ranging from 0-10 minutes for strong, or 0-30 min for moderate or weak TDIs, then

aliquots are diluted 10-20X into substrate concentrations such that velocity in the control incubations is at >80% of Vmax. High sub-strate concentration and the large dilution both significantly reduce the effects of reversible inhibition in the assay. The goal is to de-termine kobs values for each test compound concentration by plot-ting the decrease in natural logarithm of activity over time and de-termining the negative slopes of each line. Considerable debate surrounds which time points should, and should not be used for the rate determinations of kobs. Typically, the rate observed is highest at the first non-zero time point taken, especially at higher test com-pound conc., and attenuates at the longer preincubation time point(s). If the first non-zero time point is ignored, often a biphasic linearity is observed and the time points from zero to the point of that inflection are used for determining kobs. Fig. (4A) illustrates a plot of natural log of percent control activity vs. preincubation time in which thioTEPA was assessed for KI/kinact in a CYP2B6 bupropion hydroxylase experiment. The above described initially high slope can be observed at the highest thioTEPA concentration at the 2 minute preincubation time point. Also there is an apparent inflection point at 8 minutes preincubation. In this example the time points from 0 to 8 minutes were used to determine the KI/kinact val-ues shown in Fig. (4B) using the following equation:

[ ]

[ ]

[ ]IK

Ikkk

I

inactIobsobs

+

+=

=0

The kobs[I]=0 value represents the apparent inactivation observed in samples containing only vehicle (solvent) and is used so that the curve is not forced through zero, resulting in an increased KI, and decreased kinact values being calculated.

Fig. (3). CYP2B6 Bupropion hydroxylase thioTEPA IC50 plots in the pres-

ence () and absence () of 30 min NADPH preincubation. Arrow indi-cates the change in activity at the IC25 concentration.

GENERAL INDUCTION ASSESSMENT STRATEGY

Induction of P450 enzymes results in an increase in the basal level of enzyme after exposure to certain drugs. As opposed to in-hibition, induction is a slow process which is caused by a change in the balance between the normal rates of enzyme synthesis and en-zyme degradation. Either an increase in the synthesis rate or a de-crease in degradation rate will lead to an increased steady-state enzyme concentration. Induction effects are therefore observed in multidose regimens where a steady state concentration of the induc-ing drug is maintained long enough for increased enzyme levels to occur. During drug development, induction is a concern and this is reflected in the increase of induction screening earlier in compound development. This induction concern can be exacerbated by the


Fig. (4). CYP2B6 Bupropion hydroxylase thioTEPA inactivation data. Plot

of natural log percent control activity vs. preincubation time in panel A. Plot

of kobs,app vs. inactivator concentration (KI/kinact) in panel B.

possibility of a compound causing autoinduction. This is an induc-tion event whereby the compounds own metabolism is enhanced, leading to increased clearance and decreased exposure. The en-zymes CYP3A4 and CYP1A2 are predominantly affected, followed by CYP2C9 and CYP2B6 [11]. Increases in synthesis rates can result from activation of several nuclear hormone receptors: the pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR), and the constitutive androstane receptor (CAR) [55,56]. PXR binds as a heterodimer with the retinoid X receptor (RXR; CAR also utilizes this to form a heterodimer) to the PXR response element, and a distal enhancer domain upstream of the promoter has been found to be crucial to the expression of genes regulated by PXR. PXR in-duces a number of Phase I and II enzymes involved in drug metabo-lism; it is also responsible for the upregulation of many transcrip-tion factors, including AhR, CAR, and its own expression [57]. Most drugs that induce CYP3A levels are believed to do so primar-ily via PXR activation, however there is overlap in activation with CAR in the sets of target genes which are expressed (e.g., CYP2B, CYP3A, and CYP2C). This explains the overlapping CYP induc-tion patterns which exist; for example, Rifampin induces CYP2B6, CYP2C9, and CYP3A4 genes in humans [58]. The AhR receptor has been shown to control the expression of CYP1A. AhR is a ligand-activated transcription factor belonging to the family of he-lix-loop-helix DNA binding proteins [59]. The induction of CYP1A begins with the binding of an inducer to AhR, followed by ligand-dependent heterodimerization between the AhR and the AhR nu-

clear translocator (ARNT); this heterodimer then binds to the xeno-biotic response element (XRE) core sequence of 5-GCGTG-3 present in the promoter region of inducible enzymes, notably CYP1A1 and 1A2 [57].

Current Induction In Vitro Models

Animal models and animal derived reagents typically lead to poor predictions of human induction, therefore, human derived in vitro reagents are preferred [60]. A reporter gene construct contain-ing a fusion of human CYP3A4 and luciferase has been transiently transfected into HepG2 cells, and has been used to try to gain a greater understanding of the molecular mechanisms underlying the transcriptional regulation of CYP3A4 [61]. A 3 to 4-fold induction response with 5 M rifampin was seen after 48 hour treatment with the longest construct, which spanned bases -13000 to +53 of CYP3A4. A similar reporter gene construct utilizing a stable trans-fection of the same cell line, instead containing a human CYP1A fusion construct, has been used to screen AhR ligands [62]. After 28-hour treatment, 16-fold induction was achieved with the potent 1A inducer TCDD (10 nM) and 12-fold induction was obtained with 2 M of 3MC. Thus, reporter gene assays can exhibit a robust response to selected inducers and can serve as valuable screening tools. The advantages of these systems are that they are fast, rela-tively sensitive, and inexpensive to use.

El-Sankary et al. [63] also used transiently transfected HepG2 cells to generate and rank Emax and EC50 values for many well-established CYP3A4 inducers. Ripp et al. [64] expanded this ap-proach by incorporating free plasma concentrations into their Emax model to generate a relative induction score. Ripp et al. utilized the Fa2N-4 cell line, a human hepatocyte immortalized cell line using a single human donor, as a model for predicting CYP3A induction in vivo. These investigators were able to effectively generate an in vitro-in vivo robust correlation model with a large number of CYP3A4 inducers; thus, this cell line can be utilized as an effective DDI prediction tool. Youdim et al. [65] also used Fa2N-4 cells in a novel cocktail assay to measure induction of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Induction was measured by metabolic activity of enzyme-specific probes, utilizing a mass spec-trometer; significant induction of the aforementioned CYPs was achieved after a 72-hour treatment. However, induction due to CAR activation has been found to be absent with known hepatic CAR activators such as artemisinin and CITCO, 6-(4-chlorophenyl) imi-dazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime. Reduced induction was also observed for phenytoin and efavirenz which are dual activators of PXR/CAR [66].

A human hepatoma cell line, HepaRG, has been recently devel-oped which responds to PXR, CAR, and AHR activators and which has demonstrated in vitro induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 [55]. This cell line achieved significant induction, after 48-hour treatment, of CYP1A1 and CYP1A2 with omeprazole and of CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 with rifampin, utilizing RT-PCR measurement of mRNA expression. Significant induction of the aforementioned CYPs was also obtained in activity measure-ments, utilizing liquid chromatography/mass spectrometry. These investigators determined that F2 values (concentrations leading to a 2-fold increase of the baseline levels) from CYP3A4 mRNA induc-tion related to the in vivo AUC of the test compounds and could be used to correctly rank inducers and non-inducers. The investigators further suggest that F2 values could be used instead of EC50 values to assess the induction response of a cell system, especially in in-stances where an EC50 cannot be obtained due to cytotoxicity or solubility issues.

Currently, the FDA recommends using an in vitro method to evaluate a drugs potential for induction prior to in vivo studies, utilizing either freshly isolated or cryopreserved human hepatocytes [22]. While both have been used successfully with induction stud-


ies, the findings to date indicate that cryopreserved hepatocytes can be used routinely for the evaluation of drug metabolism. Li et al. [67] outlined the advantages of using cryopreserved over fresh he-patocytes as follows: ease of experimentation (use of fresh hepato-cytes involves waiting for an available donor), ease of repeat ex-perimentation, and choice of donor lots to insure adequate induction response. In addition, a complication of using fresh hepatocytes is the great variability observed between individual lots; DDIs occur-ring in the clinic due to enzyme induction can be difficult to corre-late with results seen in the general population, due to this signifi-cant intersubject variability. Since significant donor-to-donor vari-ability of induction data is common in fresh hepatocytes, it is rec-ommended that three different donors be used when attempting to predict induction in vitro. However, there is lower donor-to-donor variability when utilizing lots of cryopreserved human hepatocytes instead of fresh. Currently 3 different sources of hepatocytes, (ei-ther fresh, cryopreserved, or immortalized) are deemed sufficient for the drug development stage, while one source suffices for the discovery screening stage. Immortalized hepatocytes, despite their limitations (which include alteration of both the expression of CAR and some hepatic transporters [68] can provide a consistent re-sponse. Variations in induction of fresh and cryopreserved hepato-cytes can be minimized by normalizing to percent of the positive control (i.e., percent of rifampin for CYP3A4). As far as limitations to using cryopreserved hepatocytes vs. fresh, the aforementioned investigators found that they were minimal: a finite lifespan in sus-pension (this is offset by the availability of many lots that can at-tach, with subsequent prolonged viability), limited GSH conjuga-tion, and compromised transporter activities. The most significant development in the treatment of cryopreserved hepatocytes has been the use of a quick thaw method; cells are thawed rapidly (~1 minute) in a water bath and then poured into warmed (37C) me-dium. When compared with freshly isolated hepatocytes, cryopre-served hepatocytes have shown no relevant differences [69]. The recent availability of immortalized human hepatocyte lines and the refinement of hepatocyte cryopreservation techniques have led to a significant improvement in the availability of reagents for induction experimentation.

Handling of Cryopreserved Human Hepatocytes

In vitro incubations are typically run at 37 C in a humidified chamber under 5% CO2 in air. Three different serum-free media types are in current use with similar results: modified Chees me-dium (MCM), hepatocyte medium from Gibco (HM), and Wil-liams Medium E (WME) and all these media are typically supple-mented with insulin and dexamethasone [60,67,70]. FDA draft guidance suggests that at least three test compound concentrations be assessed with one concentration at least an order of magnitude greater than the clinical Cavg. Since in vitro induction assays are normally run in serum-free media, the fu in vitro is usually higher than that obtained in plasma [22]. Therefore, a determination of the unbound fraction of the test compound in culture media and in plasma may be desired in order to validate the drug concentrations chosen for analysis at regulatory submission [71]. Typically con-ventional monolayers of cells or sandwich cultures are utilized with cells being seeded and allowed to establish a competent monolayer during a preincubation period lasting 1-4 days; it is crucial to allow at least 24 hours for attachment, but depending on the timing of the assays, cryopreserved human hepatocytes can be plated up to four days in advance of the experiment. Since the cryopreserved cells are denser than those from cell lines, adequate mixing before and during the pipetting of the cells is critical in order to insure an equal number of cells in all wells; also, gentle swirling of the plates after seeding is needed to insure even distribution of the cells. Different lots of human cryopreserved hepatocytes have varying optimal cell densities; a variety of densities should be investigated upon evalua-tion of a new lot. Also crucial to lot evaluation for induction is the inclusion of at least 5 known inducer curves (5 to 6 conc. each)

along with suitable positive controls for CYP3A4 and CYP1A2. To assess induction, hepatocytes are typically treated with test com-pound (5-6 concentrations, triplicates per concentration) and a vehi-cle control (commonly 0.1% DMSO) for 2-3 days with a change of media/compound daily, then the activity levels are assessed using CYP selective probe substrates. Induction assessment does not cur-rently lend itself to high throughput approaches due to the long term exposure of compound needed to elicit measurable induction; typi-cally a 3-day exposure of hepatocytes to compound is required for in vitro induction studies.

Induction Activity Assays

It is believed that measurements of CYP3A and CYP1A2 activ-ity in human hepatocytes are adequate to predict drug-drug interac-tions caused by induction of drug-metabolizing enzymes. CYP3A activity is commonly measured by assessing testosterone 6-hydroxylase or midazolam 1-hydroxylase activities utilizing HPLC/UV or LC/MS/MS detection. Concentrations of 6-hydroxy-testosterone or 1-hydroxymidazolam can be extrapolated via a standard curve which contains a mixture of metabolite and its re-spective substrate. For assessing CYP1A2 activity phenacetin O-deethylation is the FDA-preferred probe substrate [22] to measure AHR-mediated induction in vitro of cryopreserved human hepato-cytes [55]. Another common endpoint for CYP1A2 activity is the O-deethylation of 7-ethoxyresorufin, which utilizes a fluorescent endpoint and quantitation of the amount of metabolite produced in each sample via a resorufin standard curve. CAR-mediated induc-tion, when assessed, is frequently monitored by following CYP2B6 mediated bupropion hydroxylase activity [33,59].

mRNA Isolation

Correlation between enzyme activity and mRNA transcription levels is expected; resultant mRNA transcription levels that are elevated in the absence of enzyme change may be indicative of concurrent induction and time-dependent inhibition, as well as to greater sensitivity of the mRNA endpoint. Total mRNA is typically extracted from cells using the mini RNeasy kit, according to in-structions provided by the manufacturer for isolation of total RNA from animal cells using a vacuum apparatus. The concentration and purity of the resultant RNA is then assessed for purity and concen-tration via spectral analysis.

mRNA Endpoints

The production of mRNA in each incubation is routinely moni-tored using commercially available 5 nuclease assay kits utilizing real-time quantitative polymerase chain reaction (RT-qPCR), or other methods, coupled with easily read fluorescent tags [72]. Both the Invader (Third Wave Technologies, Madison, WI) and TaqMan assays (Applied Biosystems, Foster City, CA) have been used to quantitatively measure CYP3A4 mRNA. The Invader assay utilizes two separate reactions. In the primary reaction, total RNA is incubated with two CYP3A4 specific-oligonucleotides and a Cleavase enzyme; the oligonucleotides bind so that there is a 1-base pair overlap, adjacent to a non-specific region of the down-stream oligonucleotide. The Cleavase enzyme removes this non-specific region, known as the 5-flap. During the secondary reac-tion, another set of two 5-flaps are added. The downstream secon-dary oligonucleotide has a FRET (Fluorescence Resonance Energy Transfer) fluorophore-quencher complex bound to its 5 end. The secondary oligonucleotides bind to the 5flap in an overlapping manner, similar to the configuration in the primary reaction. Here, the Cleavase enzyme removes the fluorophore from the oligonuce-lotide, resulting in a fluorescent signal which can be read on a plate reader; a standard curve of CYP3A4 mRNA is then used to convert fluorescence to attomoles of CYP3A4 mRNA transcript. The Taqman real-time PCR assays provide the flexibility to examine other CYP induction (CYP1A2, CYP2B6, CYP2C9, CYP2C19) besides that of CYP3A4. These assays also have the option of util-


izing a one-step or a two-step reaction, depending on assay needs. In one-step RT-PCR, the reverse transcriptase and PCR reactions take place in one buffer system for convenience. Two-step PCR involves two separate reactions: first total RNA is reverse tran-scribed into a complementary DNA strand; then this cDNA is am-plified using PCR. This method is useful for detecting multiple transcripts from a single cDNA template (i.e., examining multiple CYPs) or for storing cDNA for future use.

In quantitative real-time PCR, data are collected throughout the process; reactions are characterized by the point in time during cycling when amplification of a target is first detected, rather than the large amount of target amassed at the end of a PCR. There are two types of quantitation: absolute and relative. Absolute quantita-tion utilizes a standard curve and is most often used when it is nec-essary to determine the absolute transcript copy number [73]. Rela-tive quantitation (RQ) determines the change in expression of a nucleic acid sequence (the target) in a test sample relative to the same sequence in a sample which contains an endogenous control (such as GAPDH, glyceraldehyde-3-phosphate dehydrogenase) instead of the target probe. RQ provides an accurate comparison between the initial level of template in each sample, without requir-ing the exact copy number of the template, and does not require use of a standard curve. The amount of target, normalized to an en-dogenous reference and relative to a calibrator, is given by 2

-CT.

The derivation of this formula is described in Applied Biosystems [User Bulletin #2, P/N 4303859].

Cytotoxicity Assays

An assessment of the test compound for cytotoxicity is also recommended due to the possible combination of induction and cytotoxicity biasing the observed activities; cytotoxicity can lead to reduced mRNA levels and therefore lower fold induction results. Greater cytotoxicity is a significant limitation of cell-based assays. The following discussion includes several cytotoxicity endpoints that are available and easily measured. Adenosine tri-phosphate (ATP) is a marker of cell viability because it is present in all me-tabolically active cells and the concentration declines rapidly when the cells are about to undergo necrosis or apoptosis. The assay is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin; the emitted light is proportional to the ATP concentration, and is measured with a luminescent plate reader. The advantages to using the ATP assay are increased sensi-tivity compared with other cytotoxic assays and availability of quantitation using a standard curve [74].

WST-1 can be used for the measurement of cell proliferation and viability, based on the cleavage of the tetrazolium salt (WST-1) by mitochondrial dehydrogenases to a spectrophotometrically measurable formazan dye. A decrease in the number of viable cells results in a decrease in the overall activity of the mitochondrial dehydrogenases in the sample. This decrease in mitochondrial en-zyme activity leads to a decrease in the amount of formazan dye formed. An advantage of WST is that the end cleavage product is soluble, and does not require further mixing as compared to the MTT assay, which yields a product which must be mixed before reading [75].

The Neutral Red assay measures cell death, based on the prin-ciple that viable cells will take up the Neutral Red dye by active transport and incorporate the dye into lysosomes. Non-viable cells are defined as those which have lost their transport capabilities. An increase or decrease in the number of cells or their physiological state results in a similar change in the amount of dye incorporated into the cells; this indicates the degree of cytotoxicity caused by the test material. The dye is measured spectrophotometrically [76].

Any advantage of one system over the other for assessing cyto-toxicity is dependent upon a number of variables, including the type of cell used (primaries vs. cell lines), relative metabolic activities of

the respective cells, and the length of time the cells are exposed to compound.

Induction Data Analysis

Typically, induction results have been reported as a comparison of test compound with negative control as a fold-induction (whereby an induced activity of two fold or higher than the vehicle control was considered a positive response), or against known in-ducers such as rifampin as percent of positive control. This repre-sented a crude means of defining inductive capability, since induc-tion was measured purely by the size of the response without taking into account the concentration of compound used to generate that response; over the past several years considerable efforts have been made to move towards more classical kinetic descriptors [60]. Ross and Kenakin [77] proposed classifying drugs according to both the affinity for the receptor and efficacy once bound (comparison of EC50 values and Emax). The current 2006 draft FDA guidance now indicates that Emax, the maximum induction response, and EC50, the effective concentration at which 50% of maximal induction occurs, can now be reported in regulatory submissions[22]. Smith et al. [78] suggested that an analogy to enzyme kinetics exists, in that it is necessary to use both KM (a measure of affinity) and Vmax in order to correctly ascertain overall catalytic efficiency for turnover of a substrate. It follows then that both EC50 and Emax values are needed to adequately describe the induction efficiency of a compound. It is important to note, however, that cytotoxicity can lead to difficulty in accurately calculating the Emax values.

After a sufficient amount of treatment time has elapsed, CYP induction in an vitro model would reach a steady state. Lin [8] pro-posed that in theory, once this steady state of induction has been attained, the amount of in vivo induction (E) could be predicted for a drug candidate using the following equation, using EC50 and Emax values at the steady-state concentration of a drug [I] Fig. (5):

Fig. (5). Testosterone 6-hydroxylase rifampin induction plot (Emax/EC50). Fold-induction vs. rifampin concentration.

[ ]

[ ]IEC

IE

+

=

50

maxE

In theory, the drug concentration [I] used for predicting CYP induction should be the unbound drug concentration at the active site of the appropriate receptor (i.e., PXR, CAR, AhR). Since quan-titation of the unbound drug at the active site is not feasible, the unbound drug concentration in plasma theoretically could be used in place of the unbound drug concentration at the active site. This is


allowing for the assumption of reversible binding to plasma pro-teins, and that the drug in question crosses the cell membrane pre-dominantly by passive diffusion.

CONCLUSION

The assessment of in vitro CYP inhibition and induction during drug discovery and development is crucial to understanding the potential impact of coadministration of two or more drugs. A di-chotomy exists between the needs of the early discovery scientist who is screening thousands of NCEs and those of the late stage development scientist who is focused on a single drug candidate and accurate predictions of in vivo response. The final intended use of in vitro inhibition and induction data dictates the assay method(s) that are most appropriate. For discovery high throughput needs, some assay fidelity can be sacrificed in order to increase through-put, bearing in mind that additional study will follow. For early discovery inhibition assessments, the use of a single concentration cocktail approach using human liver microsomes offers the greatest advantage. The results generated tend to agree with those obtained using more definitive approaches, a reduced set of isoforms can be assessed for SAR, and higher throughputs can be maintained while still taking advantage of LC/MS/MS selectivity [29,49]. The need for high quality datasets which are suitable for regulatory submis-sion dictates the use of standardized methods across the pharmaceu-tical industry. For inhibition assays, this is the single probe micro-somal LC/MS/MS assay with all the appropriate standards and ana-lytical QCs, test article interference QCs, positive controls, and substrate blanks. Similarly for induction activity assessments, the analytical methodology should meet comparable standards. The methods for evaluating enzyme induction in vitro are generally as follows: use of a primary human hepatocyte model, a 2-3 day treatment period, and measurement of enzyme activities. Measure-ment of mRNA can provide useful data, given the sensitivity and flexibility of RT-qPCR technology now available. Cytotoxicity assays should be included in order to confirm questionable induc-tion results at the high end of the dose curve. Cell lines also are in use for induction screening, but the best model for predicting hu-man induction remains the use of cryopreserved or primary hepato-cytes. Cryopreserved lots of human hepatocytes have now become a readily available viable alternative to the use of freshly isolated hepatocytes for in vitro induction studies. Although the methods used for assessing CYP induction has improved significantly over the past 10 years, much remains to be learned. Due to the involve-ment of many contributing factors, quantitative prediction of CYP induction remains challenging and, similar to inhibition predictions, requires further standardized approaches to gain reproducibility between investigators. The availability of cryopreserved hepato-cytes allow for more complete characterization and effective com-parison between different lots of hepatocytes. During early drug discovery, a single source of hepatocytes can be used as a quick screen to examine a large number of compounds within a relatively short amount of time, utilizing mRNA data only. While induction is only one of many parameters examined, this information can prove useful in determining the best candidates for advancement. During the drug development stage, more in-depth induction studies (in-volving both activity and mRNA of 3A4 and other CYP isoforms - CYP1A2 and CYP2B6 for example), using three sources of hepato-cytes, can be performed. Several in vitro inhibition and induction experimental methods have been reviewed and those the authors feel best represent current best practice identified. Only by using the science which is best suited to answer the question at hand can effective decisions be made during the early discovery and devel-opment processes. Ultimately these data can be used for the quanti-tative prediction of clinical outcomes and only by maintaining a high level of confidence in the generated results can prudent deci-sions be made. The on-going assessment of new compounds for their in vitro inhibition and induction potential continues to be criti-cal component during the development of new compounds and

offers significant insight into their ability to ultimately become safe drugs.

ACKNOWLEDGEMENTS

We would like to thank Odette A. Fahmi and Drs. R. Scott Obach and Larry M. Tremaine for their critical review of the manu-script.

ABBREVIATIONS

3MC = 3-Methylcholanthrene

ADME = Absorption distribution metabolism excretion

AhR = Aryl hydrocarbon receptor

AIC = Akaike information criteria

AUC = Area under the curve

CAR = Constitutive androstane receptor

CYP = Cytochrome P450

DDI = Drug drug interaction

DNA = Deoxyribonucleic acid

DMSO = Dimethylsulfoxide

ER = Endoplasmic reticulum

FDA = Food & drug administration

IB = Investigators brochure

HPLC = High pressure liquid chromatography

IND = Investigational new drug application

LC/MS/MS = Liquid chromatography/mass spectrometry/mass spectrometry

MBI = Mechanism based inactivation

mRNA = Messenger ribonucleic acid

NADPH = Nicotinamide adenine dinucleotide phosphate (reduced)

NDA = New drug application

PCR = Polymerase chain reaction

PXR = Pregnane X receptor

RXR = Retinoid X receptor

QC = Quality control

RNA = Ribonucleic acid

TCDD = Tetrachlorodibenzodioxin

TDI = Time dependent inhibition

UV = Ultraviolet

XRE = Xenobiotic response element

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Received: May 10, 2008 Revised: July 25, 2008 Accepted: July 26, 2008

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In Vitro Cytochrome P450 Induction and Inhibition